Defining the molecular interactions between a virus and its host that regulate gene-specific transactivation has been essential to understanding DNA virus persistence and replication. The Human herpesvirus-8 (HHV-8) Rta protein is necessary and sufficient for the virus to emerge from latency and replicate (lytic reactivation). Rta interacts directly with the cellular protein called RBP-Jk isoform 1, which is also required fo lytic reactivation. RBP-Jk1 normally specifies the genes that will be activated by the cellular Notch signal transduction pathway by binding sequence specifically to DNA. In this fashion, RBP-Jk1 serves as a landing pad for the activated Notch receptor (Notch intracellular domain (NICD1)). HHV-8 Rta promotes DNA binding of RBP-Jk1 during viral reactivation, a mechanism that is fundamentally different from the canonical mechanism established for other RBP-Jk-activating proteins, NICD1 and Epstein-Barr Virus (EBV) EBNA-2. However, NICD1 induces expression of 24 HHV-8 genes without stimulating complete viral reactivation, supporting a promoter-specific mechanism for controlling its activity in HHV-8 infected cells. Recent data suggest that DNA binding of multiple RBP-Jk isoforms is regulated both positively and negatively in response to HHV-8 reactivation signals. Modulation of DNA binding of RBP-Jk is a novel level of regulation of the Notch pathway that has been underappreciated in the literature. The overall goal of this application is to define the basic molecular mechanisms that regulate RBP-Jk DNA binding in HHV-8 infected cells, and determines the differential transcriptional response of the virus to Rta or to other Notch pathway activators. Our studies will explain the fundamental regulation of productive and non-productive virus reactivation as determined by promoter-specific transactivation. We will therefore address these Specific Aims: Aim 1. To determine whether non-canonical RBP-Jk isoforms participate in Rta and NICD1 transactivation and HHV-8 reactivation. Aim 2. Identify, quantitate, and clone additional RBP-Jk isoforms from HHV8-infected B cells. A series of biochemical and molecular biological approaches are proposed. Protein-protein and protein- DNA interactions represent the basis for many of the experiments. Effects on viral reactivation will be quantitated using a novel, highly quantitative, HHV-8 reporter virus in RBP-Jk null B cells. A major part of the project involves a comprehensive assembly and quantitation of the RBP-Jk transcriptome using next generation sequencing. This proposal will shed light on how Notch target genes are specified for transactivation, and reveal new components of the Notch signal transduction pathway.